AMPK attenuates SHH subgroup medulloblastoma growth and metastasis by inhibiting NF-κB activation

Background Medulloblastoma (MB) is one of the most common malignant pediatric brain tumors. Metastasis and relapse are the leading causes of death in MB patients. The initiation of the SHH subgroup of MB (SHH-MB) is due to the aberrant activation of Sonic Hedgehog (Shh) signaling. However, the mechanisms for its metastasis are still unknown. Results AMP-dependent protein kinase (AMPK) restrains the activation of Shh signaling pathway, thereby impeding the proliferation of SHH-MB cells. More importantly, AMPK also hinders the growth and metastasis of SHH-MB cells by regulating NF-κB signaling pathway. Furthermore, Vismodegib and TPCA-1, which block the Shh and NF-κB pathways, respectively, synergistically restrained the growth, migration, and invasion of SHH-MB cells. Conclusions This work demonstrates that AMPK functions through two signaling pathways, SHH-GLI1 and NF-κB. AMPK-NF-κB axis is a potential target for molecular therapy of SHH-MB, and the combinational blockade of NF-κB and Shh pathways confers synergy for SHH-MB therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-023-00963-2.


Introduction
Medulloblastoma (MB) represents the most frequent central nervous system (CNS) malignant tumor in pediatrics and an embryonic tumor originating in the cerebellum [1,2]. Four distinct molecular subgroups of MB termed Wingless (WNT), Sonic hedgehog (SHH), Group 3, and Group 4 were identified based on transcriptional profiling of large cohorts [3][4][5]. Relapses occur in 30% MB patients after surgery, and the metastasis rate and the 3 years survival rate of relapsed patients are 86% and 18% [6]. Metastatic cells detach from primary tumors and disseminate to the leptomeninges by passive spread through the cerebrospinal fluid or to the brain or other organs through a hematogenous route [7,8]. Although the molecular mechanisms of MB primary tumors are well elucidated, the molecular defects underlying metastasis are still unclear.
SHH-MB arises from granule cell precursors in the developmental cerebellum following the aberrant activation of the Shh pathway [9]. Binding the secreted hedgehog ligand Shh to the membrane receptor Patched-1 (Ptch1) relieves the inhibitory effect of Ptch1 on the downstream G protein-coupled receptor Smoothened (Smo) and turns on the signaling events leading to the activation of transcription factor Gli1 [10]. Despite Smo inhibitor Vismodegib (GDC-0449), which FDA approves, having short-term effects for MB in clinical trials, drug resistance is frequently developed and eventually results in treatment failure [11][12][13]. Resistance mechanisms in MB may include mutations in drug-targeting molecules, new mutations downstream, and activation of other pathways in MB progress [14,15]. To control the progress and relapse of MB, it is urgent to fully understand the specific metastasis mechanism of MB and find new molecular targets for that.
The AMP-activated protein kinase (AMPK), as an essential sensor of cellular energy status, can phosphorylate multiple substrates to respond to low energy levels and regulate cell metabolism and growth. AMPK is a heterotrimeric complex comprises catalytic α subunits (α1, α2), regulatory β (β1, β2) and γ subunits (γ1, γ2, γ3), which is activated by the phosphorylation of T172 site of AMPKα subunit [16]. AMPK has dual effects in different cancers, promoting and inhibiting tumor progress depending on its downstream substrates [17]. In Shh pathway, AMPK can interrupt GLI1 transcriptional activity through the phosphorylation of GLI1 at serines 102 and 408 and threonine 1074 [18,19]. However, it is unknown whether the expression of AMPK subunits is abnormal in MB, and the mechanism of AMPK in the growth and metastasis of MB is not completely clear.
Here, we found that AMPK inhibited the proliferation and metastasis of SHH-MB cells by restraining the activation of Shh and NF-κB pathways. The combination of blocking NF-κB and Shh pathway had a synergically therapeutic effect on SHH-MB, revealing the AMPK-NF-κB axis as a potential target for SHH-MB treatment.

Expression of PRKAA1 in MB is lower than in the normal cerebellum
To identify the connection between AMPK and MB progression, we first performed immuno-histochemical staining of normal cerebellum and MB tissues. 2 in 5 SHH-MBs and 5 in 6 Group 4 MBs showed significantly reduced signal of phosphorylated AMPKα compared to normal cerebellum (Fig. 1A). The differential mRNA expression analysis between MB and normal cerebellum using the MB patient dataset (GSE124814) found that PRKAA1 (AMPKα1) mRNA expression was downregulated in MBs (Fig. 1B). These results suggest that activated AMPK is reduced in MB tissues. PRKAA1 mRNA expression was not consistent in four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4), which was significantly decreased in Group 4 MB tissues, but slightly increased in SHH-MB compared to normal (Fig. 1C). Kaplan-Meier survival analysis showed that the patients with higher PRKAA1 expression had longer survival durations than those with lower PRKAA1 expression (Fig. 1D). The positive correlation between PKRAA1 levels and survival time was shown in all four subgroups of MB patients. These results suggest that high-level expression of PRKAA1 may be a sign of favorable prognosis for MB patients.
It was noted that there was no mutation in several genes denoting AMPK subunits reported in MB tissue from The Cancer Genome Atlas (TCGA) cohort, although CTNNB1, PTCH1, and TP53 genes were missense-mutated or deleted in 5-10% cases (Fig. 1E). It suggested that repressed transcription and activation of AMPKα in MB tissues were likely due to epigenetic aberrations.

AMPK suppresses DAOY cell growth
Since the SHH subtype of MB has an ostensibly simple tumor origin and is well-studied, the SHH subtype of MB cell line DAOY was selected for our further study. To determine whether AMPKα controls MB cells' growth and malignant progression, we used small interfering RNA (siRNA) to knock down PRKAA1 expression in DAOY cells. The knockdown efficiency of siPRKAA1 was validated by Western Blot (Additional file 1: Fig S1A). Knockdown of PRKAA1 promoted the proliferation of DAOY cells according to CCK-8 assay (Additional file 1: Fig S1B) and EdU incorporation assay (Additional file 1: Fig S1C, D). Besides, siRNA targeting PRKAA1 encouraged DAOY cells to form colonies (Additional file 1: Fig S1E, F). In addition, flow cytometric analysis revealed that siRNA against PRKAA1 significantly increased the proportion of cells in the S-phase and decreased the proportion of cells in the G1-phase (Additional file 1: Fig S1G, H). Moreover, according to the CCK-8 assay, knockdown of PRKAA1 also promoted the proliferation of D283 Med, a representative Group 3/4 subgroup MB cell line (Additional file 1: Fig S1I, J). Next, to eliminate the off-target effects of siRNA, we used CRISPR/Cas9 genome editing technology to generate PRKAA1 −/− DAOY cells. Two short guide RNAs were designed to target sites at exons 8 and 10 of PRKAA1 gene, and a 2062 bp deletion was induced in the DAOY genome (Additional file 2: Fig S2A). The knockout of PRKAA1 was verified by Sanger sequencing and Western blot (Additional file 2: Fig S2A, Fig. 2A). The proliferation ability of PRKAA1 −/− DAOY cells was enhanced compared with PRKAA1 +/+ cells shown by the CCK-8 assay (Fig. 2B). Moreover, the introduction of exogenous PRKAA1 gene compromised the proliferation ability of PRKAA1 −/− DAOY cells (Fig. 2C, D). These results indicated that AMPK controls the proliferation of DAOY cells.
DAOY cells could form tumorsphere in 3D culture, suggesting their characteristics of cancer stem cells. The self-renewal capability was increased, as assessed by extreme limiting dilution assays, following the deletion of PRKAA1 in DAOY cells (Fig. 2E). PRKAA1 −/− cells generated higher numbers of tumorspheres than PRKAA1 +/+ cells (Fig. 2F, G). In brief, we found that AMPK limits the self-renewal ability of DAOY cells.
To determine the contribution of AMPK to tumor growth in vivo, we monitored the growth of PRKAA1 −/− DAOY cells in the cerebellum of NOD-SCID mice. PRKAA1 −/− cells and their PRKAA1 +/+ control were infected with lentiviral particles to express Firefly Luciferase (Luc) gene stably and subsequently referred to as PRKAA1 −/− -Luc and PRKAA1 +/+ -Luc cells. These cells were then implanted into the cerebella of NOD-SCID mice. Animals were subjected to weekly in vivo bioluminescent imaging (BLI) to monitor the orthotropic tumor growth (Fig. 2H). Serial BLI images showed that xenografts from PRKAA1 −/− -Luc cells grew faster than those from control cells (Fig. 2I). Then the animals were sacrificed, and the cerebellum tissues were collected for histological analysis. HE staining of cerebellum tissues confirmed the presence of xenografts in the cerebellum (Fig. 2J). PRKAA1 −/− MB lesions had more Ki67-positive cells, indicating enhanced proliferation ability (Fig. 2K). Subcutaneous xenografts in nude mice provided additional evidence for the growth-suppressing effect of AMPK (Additional file 2: Fig S2B). PRKAA1 −/− xenografts exhibited significantly increased tumor volumes, weights, and the percentage of Ki67-positive cells (Additional file 2: Fig S2C-E). Taken together, these results suggest that AMPK could hamper the growth of SHH subgroup MB cells.

AMPK controls DAOY cell migration and invasion
We noticed that introduction of siPRKAA1 changed the morphology of DAOY cells, in addition to their proliferation capacity. The DAOY cells carrying siPRKAA1 showed an elongated spindle shape rather than the polygonal shape of the control DAOY cells carrying siNC in culture in vitro (Additional file 3: Fig  S3A). By Phalloidin labeling, we found that the control cells showed diffusely distributed actin filaments (F-Actin) and filopodia-like protrusions. In contrast, PRKAA1 knockdown cells displayed significant aggregation of F-actin bundles and smooth lamellipodialike edges (Additional file 3: Fig S3A). Furthermore, we performed transwell assays to determine the migratory and invasive capacities of DAOY cells. Knockdown of PRKAA1 significantly enhanced the migratory and invasive abilities of DAOY cells (Additional file 3: Fig  the decreased expression of epithelial cell marker E-cadherin and increased expression of mesenchymal cell markers N-cadherin and MMP3 versus control PRKAA1 +/+ cells ( Fig. 3K and Additional file 3: Fig S3H). siPRKAA1 also promoted the EMT process by showing increased levels of mesenchymal cell markers (Additional file 3: Fig S3I). To determine whether PRKAA1 −/− DAOY cells exhibited increased metastatic potential in vivo, we separately transplanted PRKAA1 −/− -Luc and PRKAA1 +/+ -Luc DAOY cells into mouse cerebella and monitored the leptomeningeal metastasis seven weeks after implanting. PRKAA1 −/− cells produced spinal metastases based on the bioluminescent imaging and pathological examination ( Fig. 3L-N).

AMPK-GLI1 inhibits DAOY cell growth but not metastasis
Although it was reported that activation of AMPK suppresses MB cell growth through inhibition of GLI1 activity and expression by phosphorylating and destabilizing GLI1 protein [20], the molecular mechanism by which AMPK inhibits DAOY cells proliferation and metastasis remains not entirely clear. Since SHH-MB is characterized by an overall deregulation of SHH pathway, we performed a western blot analysis to examine the levels of transcription factors GLI1-3 (Fig. 4A). As expected, GLI1 accumulated at a higher level PRKAA1knockdown DAOY cells, While the levels of GLI2, GLI3 activator and repressor were not changed (Fig. 4A). To investigate whether the accumulated GLI1 contribute to the overgrowth and metastasis of DAOY cells driven by AMPKα loss, we performed a double-knockdown of both PRKAA1 and GLI1 in DAOY cells (Fig. 4B). Significantly, GLI1 depletion abolished accelerated cell proliferation in PRKAA1 knockdown cells, as illustrated by CCK-8 assay (Fig. 4C). The flow cytometric proliferation assay confirmed that GLI1 depletion rescued PRKAA1 deficiency-induced accumulation of cells in S-phase (Fig. 4D, E). These results demonstrated that the accumulation of GLI1 is responsible for the accelerated proliferation of DAOY cells seen upon PRKAA1 loss, which is consistent with the literature reports. However, siGLI1 did not counteract the enhanced migration of DAOY by siPRKAA1 in the transwell migration assay (Fig. 4F, G), suggesting that AMPK hampered DAOY migration through some unknown signaling pathways except for the SHH-GLI1 pathway.

AMPK restrains DAOY cell metastasis through inhibiting NF-κB pathway
To further elucidate the underlying mechanism that AMPK inhibits the proliferation and metastasis of DAOY, we performed transcriptome sequencing in DAOY depletion of PRKAA1. Knockdown of PRKAA1 induced significant gene expression changes in DAOY cells. The volcano plot depicted that 334 genes were up-regulated and 317 were down-regulated (Fig. 5A). The KEGG pathway analysis showed the enrichment for NF-κB pathway (Fig. 5B). The gene set enrichment analysis (GSEA) and a hierarchical clustering heatmap analysis also revealed a significant activation of NF-κB pathway in PRKAA1 knockdown DAOY cells (Fig. 5C, D). The effects of AMPK depletion on activating NF-κB signal pathway transduction were validated by real-time qPCR of CCL3, CCL5, CXCL10, and VCAM1, the downstream target genes of the NF-κB pathway (Fig. 5E). Moreover, the introduction of exogenous PRKAA1 gene reduced the transcripts of those target genes in PRKAA1 −/− cells (Fig. 5F). Activation of NF-κB pathway occurs after the degradation of pathway inhibitor IκBα and the phosphorylation of NF-κB p65. Western blots showed that the phosphorylation of P65 was increased in cells knockdown of PRKAA1, companying with the decrease of IκBα, which suggested the activation of the NF-κB signal pathway. Meanwhile, similar results existed in PRKAA1 −/− cells and were reversed by the introduction of exogenous PRKAA1 cDNA (Fig. 5G). It is noticed that the total levels of P65 was induced in PRKAA1 −/− cells without enhanced transcription of RELA (data not shown), suggesting that the degradation of P65 is impaired in PRKAA1 −/− cells. The above-mentioned results indicate that AMPK inhibits NF-κB signaling in DAOY cells.
To determine AMPK inhibits the proliferation and metastasis of DAOY through NF-κB signaling, we performed a double-knockdown of both PRKAA1 and CCL5 in DAOY cells (Fig. 5H). Notably, CCL5 depletion slowed down the accelerated cell proliferation induced by AMPK knockdown (Fig. 5I). More importantly, the enhanced migration and invasion ability of AMPK knockdown cells were significantly weakened by siCCL5 (Fig. 5J-M). Overall, these results demonstrated that AMPK attenuated DAOY cell growth and metastasis through inhibiting NF-κB pathway.

Discussion
Medulloblastoma (MB) is a highly malignant pediatric brain tumors (WHO grade IV), metastasis and recurrence are the main causes of death in MB patients [23]. SHH signaling plays an indispensable role during embryonic development, and aberrant activation of Shh pathway drives the origination of SHH subgroup MB, accounting for nearly one-third of MB cases [24]. Although the genetic divergence driving the primary tumorigenesis is commonly conserved at metastasis, the gene expression patterns of metastatic tumors remain distinct from non-metastatic primary tumors. Here, we report that lower expression of PRKAA1 predicts poor prognosis of MB, and loss of AMPK promotes the growth, stemness and metastasis of SHH-MB cells. AMPK phosphorylates GLI1, then expedites the proteasome-mediated degradation of GLI1 by promoting the binding of GLI1 with its E3 ligase β-TrCP. This inhibitory activity of AMPK in Shh signaling partially explains the accelerated proliferation of SHH-MB cells lacking AMPKα. NF-κB is one of the nuclear transcription factors function in a variety of biological processes, which exists in almost all types of mammalian cells. In most cancers, the activation of NF-κB is enhanced due to increased stimuli of the NF-κB pathway, such as increased TNFα and IL-1 in the tumor microenvironment, which can accelerate cell proliferation, inhibit apoptosis, promote cell invasion and metastasis, and stimulate angiogenesis and elongation [25]. Genetic deletions affecting regulators of the NF-κB pathway, including NFKBIA and USP4, were identified in Group 4 MB [26]. Our study elucidated that AMPK attenuates further progress of SHH-MB by inhibiting NF-κB activation, slowing down cell proliferation, and arresting migration and invasion. Cerebrospinal fluid (CSF) is the window into the central nervous system, through which MB invade and disseminate to the spinal cord [27]. Although we could not collect enough cerebrospinal fluid from mice with intracranial tumor metastasis, recent clinical research analyzed the RNA-sequencing and high-resolution mass spectrometry data of CSF samples from patients with or without MB and reported differentially expressed genes enriched in TNF-α signaling via NF-κB, which supports our results strongly [27]. Overall, our results demonstrate that AMPK attenuates the growth and metastasis of SHH-MB by inhibiting NF-κB activity. Elucidating the mechanism of MB metastasis provides the opportunity to prevent its metastasis and to reduce the mortality of MB patients. In recent years, FDAapproved Smo antagonists Vismodegib and Sonidegib were available for SHH-driven tumors, but drug resistance and relapse have also appeared in clinical use [28]. Metformin and A769662 activate AMPK, then inhibit GLI1 activity to restrain MB cells growth in vitro and in vivo, thereby sensitizing SHH-MB to Vismodegib and overcoming Vismodegib-resistance [20]. However, AMPK agonists may not be effective in metastatic MB due to the low expression of AMPKα. So we sought to explore the novel molecular targets of MB therapy at the downstream of AMPK. AMPK-NF-κB axis functions in the metastasis of SHH-MB. There have been thousands of molecules described as capable of interfering with the NF-κB signaling directly or indirectly [25]. TCPA-1 is one of them interfering with the activation of NF-kB by selectively inhibiting IKK-2. It has been used in tumor therapy trials [29,30]. Based on these considerations, we combined SHH and NF-κB inhibitors Vismodegib and TPCA-1 to treat SHH-MB lacking AMPKα. Our results represented that these two drugs synergistically restrain the growth, migration, and invasion of SHH-MB cells in vitro, suggesting that therapies might be efficacious for SHH-MB at metastasis and recurrence. It is necessary to conduct more research in the future to clarify the importance of activating AMPK and inhibiting GLI1 and NF-κB pathways for the prevention of metastasis in an in vivo model to determine whether they have clinical translational value.

Conclusions
This study demonstrated that AMPK functions as a tumor suppressor in MB progression through two signaling pathways, SHH-GLI1 and NF-κB (Fig. 7). NF-κB inhibitor and Shh inhibitor synergistically suppress MB cell growth, revealing the AMPK-NF-κB axis as a potential target for molecular therapy of SHH-MB. The anti-cancer effect of a combination of Vismodegib and TPCA-1 was superior to Vismodegib alone in MB targeted therapy (Fig. 7). Future studies are required to ascertain whether these two drugs have synergistically therapeutic effects in vivo.

Immunohistochemical staining
Medulloblastoma tissues obtained from Children's hospital of Nanjing Medical University (Nanjing, China) were carried out to detect the phosphorylation of AMPKα by immunohistochemistry. Mouse heterograft tumor tissues were detected Ki67 to measure the growth of heterograft tumors. And standard procedures for immunohistochemical staining were followed with rabbit two-step detection kit (ZSGB-BIO). The primary antibodies were rabbit anti-phospho-AMPKα (Cell Signaling Technology; 1:100), rabbit anti-Ki67 (Abcam; 1:100).

EdU incorporation assays
Transfected cells were seeded at 5 × 10 4 cells/well in 24-well plates containing round coverslips and maintained in medium overnight. Cell proliferation was further evaluated through measuring the incorporation of EdU with EdU Cell Proliferation Assay Kit (RiboBio). Images were captured by using a fluorescence microscope, and the proliferating cells in five different fields were counted.

Colony formation assays
For the colony formation assay, transfected cells were plated in triplicate at 200 cells per well in 6-well plates and cultured for 2 weeks. The cells were fixed with 4% paraformaldehyde for 15 min at 4 ℃, and then stained with 0.1% crystal violet for 30 min, colonies with more than 50 cells were counted.

Flow cytometric analysis for cell cycle
Cells were inoculated in 6-well plates and transfected with siRNA for 48 h, then these cells were collected

CRISPR-Cas9 genome editing
Genome editing was achieved using the CRISPR-Cas9 technique in DAOYs. Briefly, a single guide RNA (sgRNA) targeted the eighth and tenth exon of human PRKAA1 was designed and cloned into pX330 vector (RRID: Addgene_101733). The sgRNA sequences are: 5′-CAC CGA ACT ATA TGA TGG ATC CTC -3′; 5′-CAC CGC AAC TAT CGA TCT TGC CAA -3′. Cells were co-transfected with CRISPR/Cas9 plasmids and empty plasmids containing puromycin resistance genes. 48 h after transfection, transfected cells were selected using Puromycin (3 μg/ml) for 7 days, followed by another 4 days without selection for expansion. Then the survived DAOYs were seeded at 1 cell per well in 96-well plates for single cell colonies formation and screened by genotyping and Western Blot.
In vivo xenograft tumor model 6 weeks female BALB/c nude mice were purchased from Nanjing Medical University Experimental Animal Center. Nude mice were subcutaneously inoculated with 5 × 10 6 PRKAA1 +/+ or PRKAA1 −/− DAOY cells in double side axillae. The tumor sizes were measured every week. The tumor volume was calculated as follows: volume (mm 3 ) = length (mm) × width (mm) 2 /2. 8 weeks after xenograft inoculation, mice were euthanized and their tumor nodules were excised, photographed, weighed and harvested.

Intracranial brain tumor xenografts
NOD-SCID mice were anesthetized, and an incision was made to expose the skull. A hole was created in the calvarium above the right cerebellar hemisphere, where 2 mm lateral (right) to the sagittal suture, and 2 mm posterior of the lambdoid suture, using a microdrill. Orthotopic MB xenografts were performed by injection of PRKAA1 +/+ DAOY-Luc and PRKAA1 −/− DAOY-Luc cells severally, that had been previously infected with lentiviral particles to stably express luciferase gene, into the cerebellum of five NOD-SCID mice. Bioluminescence imaging was performed weekly using IVIS Spectrum (Perkin Elmer). Tumor growth was evaluated by quantifying the bioluminescence signals using the integrated fluxes of photons within each area of interest using the Living Images Software Package 4.5 (Perkin Elmer). All mice were euthanized when the mice showed symptoms of metastasis.

Movement of living cells
Cells were seeded in 60-mm plate at 30% density for 12 h and then treated with 2 μg/ml mitomycin C for 24 h. After, cells were cultured with fresh culture medium and recorded at one image every 5 min with Celldiscoverer 7 automatic live cell imaging system (ZEISS) for 20 h. The cells motion trajectory image and data were obtained using ImageJ (RRID:SCR_003070).

Cytoskeleton staining
Transfected cells were seeded on glass coverslips for 24 h and fixed with 4% PFA for 20 min at 4℃, then the cells were permeabilized with 0.5% TritonX-100 for 10 min at room temperature. Fluorescently labeled phalloidin working solution was added a well and incubated at room temperature for 30 min for staining and stained with DAPI. The cell cytoskeleton images were acquired with laser scanning confocal microscope.

In vitro migration and invasion assays
The 24-well plate with 8 µm pore polycarbonate membrane inserts (Millipore) was used to analyze the migratory abilities of tumor cells and then the membrane was coated with 60μL diluted Matrigel (1:30; Corning) to detect cells' invasion. After adding 600μL 10% FBS medium into the lower chambers, 2.5 × 10 4 transfected cells in 300μL serum-free medium were seeded into the insert for incubation at 37 °C in 5% (v/v) CO 2 incubator for 16 h. Then, the cells migrating to the lower surface of the membrane insert were stained with the crystal violet (Beyotime) and quantified by counting five randomly chosen microscopic fields.

RNA sequencing
Total RNA was isolated with RNAiso Plus reagent (TaKaRa) from DAOY cells transfected with siNC or siPRKAA1 for 48 h, then purified to meet the following requirements were used in subsequent experiments: RNA integrity number (RIN) > 7.0 and a 28S:18S ratio > 1.8. The triplicate samples of both assays were constructed an independent library, and sequenced on an Illumina Novaseq 6000 sequencer by CapitalBio Technology (Beijing, China). The sequencing raw data was uploaded to the GEO dataset (GSE218076).

Reverse transcription (RT) and real-time PCR
Total RNAs were isolated from cultured cells and reverse transcribed using HiScript II Q RT SuperMix (Vazyme). Quantitative real-time PCR (qPCR) was carried out using AceQ qPCR SYBR Green Master Mix (Vazyme). Each measurement was repeated three times, and each sample was analyzed in triplicate with hypoxanthine phosphoribosyl transferase (HPRT) as an internal control. The qPCR primers are listed: Human To understand the Combination Index (CI), cells were treated with Vismodegib (GDC-0449) alone, or TPCA-1 alone, or a combination of both in the following concentrations (0, 1/4 IC 50 , 1/2 IC 50 , IC 50 , 2 IC 50 , and 4 IC 50 ). To test for synergy, and then the CCK8 data was validated using CompuSyn software based on Chou-Talalay quantitative method. The dose-effect curve was simulated and CI was calculated at a constant ratio of 1:2.